This renewal application proposes to examine two fundamental issues in hematopoietic stem cell (HSC) biology, namely the relationship between cell cycle status of these cells and maintenance of their hematopoietic potential and how the dynamics between mitotic quiescence and HSC potential change during hematopoietic development. In the previous funding period, we surveyed the hematopoietic potential of cycling and quiescent HSC from human hematopoietic tissues through ontogeny and established that in adult tissues, a hierarchy of hematopoietic potential can be assembled based on the mitotic status of HSC. However, this hierarchical order does not predominate during fetal development where mitotically quiescent and cycling HSC, sustain long-term hematopoiesis. We examined if extrinsic factors can modulate the fate of primitive hematopoietic progenitors and determined that, with appropriate intervention, hematopoietic function and self-renewal can be maintained through successive in vitro divisions. These modulators included unidentified factors from committed progeny of HSC thus corroborating our proposed instructional clonal activation as a model for maintenance of steady state hematopoiesis and the stem cell pool. New information gathered to date, prompt us to pursue new and challenging concepts regarding our ability to extrinsically modulate HSC fate and the outcomes of the interaction between stem cells and their hematopoietic microenvironment at different stages of development. To better understand these critical issues in stem cell biology, two specific aims will be examined. First we will investigate if microenvironmental cues in fetal but not adult hematopoietic tissues induce dividing stem cells to retain their reconstitution potential while traversing through all phases of cell cycle. Second, we will investigate if stem cell fate can be modulated by factors elaborated by different hematopoietic and non-hematopoietic cell types including endothelial and myo-epithelial cells and more differentiated, lineage committed hematopoietic cells. Testing these specific aims should yield valuable information regarding normal hematopoiesis and the mechanisms that regulate stem cell fate. These studies are critical for assessing the impact of the interaction of stem cells with their hematopoietic microenvironment on stem cell function throughout ontogeny.